Plasmid location of Borrelia purine biosynthesis gene homologs

Abstract

The Lyme disease spirochete Borrelia burgdorferi must survive in both its tick vector and its mammalian host to be maintained in nature. We have identified the B. burgdorferi guaA gene encoding GMP synthetase, an enzyme involved in de novo purine biosynthesis that is important for the survival of bacteria in mammalian blood. This gene encodes a functional product that will complement an Escherichia coli GMP synthetase mutant. The gene is located on a 26-kb circular plasmid, adjacent to and divergent from the gene encoding the outer surface protein C (OspC). The guaB gene homolog encoding IMP dehydrogenase, another enzyme in the purine biosynthetic pathway, is adjacent to guaA. In Borrelia hermsii, a tick-borne relapsing fever spirochete, the guaA and guaB genes are located on a linear plasmid. These are the first genes encoding proteins of known function to be mapped to a borrelial plasmid and the only example of genes encoding enzymes involved in the de novo purine biosynthesis pathway to be mapped to a plasmid in any organism. The unique plasmid location of these and perhaps other housekeeping genes may be a consequence of the segmented genomes in borreliae and reflect the need to adapt to both the arthropod and mammalian environments.

A family of genes located on four separate 32-kilobase circular plasmids in Borrelia burgdorferi B31

“…Several observations suggest that the small DNA species of B. burgdorferi and other spirochetes may be fundamentally different from the plasmids of other bacteria, the most significant being that there are linear plasmids in B. burgdorferi (4,5,7,13,22,23,42). Also, two B. burgdorferi genes involved in purine biosynthesis are located on a circular plasmid (29), which appears to be inconsistent with the classical definition of a plasmid as a nonessential extrachromosomal element. Other plasmids of B. burgdorferi appear to be capable of undergoing recombination to form multimeric plasmids (25,31), and there have been reports of DNA sequences located on several different plasmids within the same bacterium (46,47,(54)(55)(56).…”

“…The filter was stripped and rehybridized with a probe specific for guaA, which is located on the same 26-kb circular plasmid as ospC (28,29,39). This probe hybridized to a linear molecule of approximately 26 kb and to two circular DNAs (Fig.…”

“…Following electrophoresis in the second dimension, the gel was soaked in several changes of water, and DNA was visualized by ethidium bromide staining. DNA was transferred to nylon membranes (ICN) and hybridized with radiolabelled DNA probes at 55ЊC as previously described (29). Filters were washed with 0.2ϫ SSC-0.1% SDS at 55ЊC, and specifically bound DNAs were detected by autoradiography.…”

“…by random priming (Life Technology, Gaithersburg, Md.) and used to screen a B. burgdorferi B31 DNA library in Lambda ZAPII (Stratagene, La Jolla, Calif.) (29). LambdaZAPII clones that hybridized with this probe were excised in vivo to produce plasmids by following the manufacturer's instructions, and the inserts were sequenced.…”

“…The digested DNAs were separated on a 0.7% agarose gel by pulsed-field electrophoresis for 18 h at 7 V/cm with program 3 of a PPI-200 programmable power inverter (MJ Research, Watertown, Mass.). DNAs were transferred bidirectionally to nylon membranes (ICN, Irvine, Calif.) and hybridized (29) at 55ЊC with a probe specific for each erp locus. Filters were washed with 0.2ϫ SSC (1ϫ SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate (SDS) at 55ЊC, and specifically bound DNAs were detected by autoradiography.…”